Chemiluminescence sensor array

ABSTRACT

Embodiments of the invention relate to integrated chemiluminescence devices and methods for monitoring molecular binding utilizing these devices and methods. These devices and methods can be used, for example, to identify antigen binding to antibodies. The devices include both a chemiluminescence material and a detector integrated together.

FIELD OF INVENTION

The embodiments of the invention relate to integrated chemiluminescencedevices and methods for monitoring molecular binding utilizing thesedevices and methods. These devices and methods can be used, for example,to identify antigen binding to antibodies. The invention transcendsseveral scientific disciplines such as polymer chemistry, biochemistry,molecular biology, medicine and medical diagnostics.

BACKGROUND

FIG. 1 shows a typical “sandwich” assay for identifying and quantifyingan antigen. This is called a sandwich assay because it uses 2 or moreantibodies to sandwich the antigen. In the typical sandwich assay aprimary antibody is bound to a support substrate. This primary antibodyis used as a capture antibody to capture a specific antigen in solution.A second antibody that attaches to the same antigen as the primaryantibody is then added. This second antibody includes a tag of some sortfor identifying the presence of the second antibody attached to theantigen. This tag may include, for example, a complex that fluoresceswhen exposed to laser light. This fluorescent signal can then bedetected by a separate light detector.

One Example of a sandwich assay is an Enzyme-Linked Immunosorbent Assay(ELISA or EIA for short). The ELISA assay utilizes two antibodies, oneof which is specific to the antigen and the other of which is coupled toan enzyme. This second antibody gives the assay its “enzyme-linked”name. The enzyme promotes a reaction involving an enzyme convertedsubstrate, which produces a signal that can be proportional to theamount of the antigen that is present.

One drawback of the sandwich assay technique is the number of steps thatare typically required to carry out the technique. For example, at leastthe following steps are typically performed: 1) Prepare a surface plateto which a known quantity of antibody is bound; 2) Apply theantigen-containing sample to the plate; 3) Wash the plate, so thatunbound antigen is removed; 4) Apply the enzyme-linked antibodies whichare also specific to the antigen; 5) Wash the plate, so that unboundenzyme-linked antibodies are removed; 6) Apply a chemical which isconverted by the enzyme into a chemical that can generate fluorescent orcolor signal; and 7) View the result: if it fluoresces, then the samplecontained antigen.

In addition to the number of steps, several separate specializedcompositions and devices are used in the sandwhich assay technique. Forexample, two different antibodies are used and a separate detector fordetecting the fluorescence is used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sandwich assay for identifying and quantifying anantigen.

FIG. 2 shows an embodiment of an integrated chemiluminescence device anddetector according to this invention.

FIG.3 shows an embodiment of an integrated chemiluminescence device anddetector including an antigen according to this invention.

DETAILED DESCRIPTION

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “an array” may include a plurality ofarrays unless the context clearly dictates otherwise.

An “array,” “macroarray” or “microarray” is an intentionally createdcollection of molecules which can be prepared either synthetically orbiosynthetically. The molecules in the array can be identical ordifferent from each other. The array can assume a variety of formats,e.g., libraries of soluble molecules; libraries of compounds tethered toresin beads, silica chips, or other solid supports. The array couldeither be a macroarray or a microarray, depending on the size of thesample spots on the array. A macroarray generally contains sample spotsizes of about 300 microns or larger and can be easily imaged by gel andblot scanners. A microarray would generally contain spot sizes of lessthan 300 microns. A multiple-well array is a support that includesmultiple chambers for containing sample spots.

“Solid support,” “support,” and “support substrate” refer to a materialor group of materials having a rigid or semi-rigid surface or surfaces.In some aspects, at least one surface of the solid support will besubstantially flat, although in some aspects it may be desirable tophysically separate synthesis regions for different molecules with, forexample, wells, raised regions, pins, etched trenches, or the like. Incertain aspects, the solid support(s) will take the form of beads,resins, gels, microspheres, or other geometric configurations. Thesupport may be made from a variety of materials including glass, nickel,magnetic metal, gold, silicon, nitrocellulose, or PolyvinylideneDifluoride (PVDF).

The term “analyte”, “target” or “target molecule” refers to a moleculeof interest that is to be analyzed and can be any molecule or compound.Some examples of analytes may include a small molecule, biomolecule, ornanomaterial such as but not necessarily limited to a small moleculethat is biologically active, nucleic acids and their sequences, peptidesand polypeptides, as well as nanostructure materials chemically modifiedwith biomolecules or small molecules capable of binding to molecularprobes such as chemically modified carbon nanotubes, carbon nanotubebundles, nanowires, nanoclusters or nanoparticles. The analyte moleculemay be fluorescently labeled DNA or RNA.

An analyte can be in the solid, liquid, gaseous or vapor phase. By“gaseous or vapor phase analyte” is meant a molecule or compound that ispresent, for example, in the headspace of a liquid, in ambient air, in abreath sample, in a gas, or as a contaminant in any of the foregoing. Itwill be recognized that the physical state of the gas or vapor phase canbe changed by pressure, temperature as well as by affecting surfacetension of a liquid by the presence of or addition of salts etc.

The analyte can be comprised of a member of a specific binding pair(sbp) and may be a ligand, which is monovalent (monoepitopic) orpolyvalent (polyepitopic), usually antigenic or haptenic, and is asingle compound or plurality of compounds which share at least onecommon epitopic or determinant site. The analyte can be a part of a cellsuch as bacteria or a cell bearing a blood group antigen such as A, B,D, etc., or an HLA antigen or a microorganism, e.g., bacterium, fungus,protozoan, or virus. In certain aspects of the invention, the analyte ischarged.

A member of a specific binding pair (“sbp member”) is one of twodifferent molecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand) or analyte and probe. Therefore, a probe is amolecule that specifically binds an analyte. These will usually bemembers of an immunological pair such as antigen-antibody, althoughother specific binding pairs such as biotin-avidin, hormones-hormonereceptors, nucleic acid duplexes, IgG-protein A, polynucleotide pairssuch as DNA-DNA, DNA-RNA, and the like are not immunological pairs butare included in the invention and the definition of sbp member.

Specific binding is the specific recognition of one of two differentmolecules for the other compared to substantially less recognition ofother molecules. Generally, the molecules have areas on their surfacesor in cavities giving rise to specific recognition between the twomolecules. Exemplary of specific binding are antibody-antigeninteractions, enzyme—enzyme converted substrate interactions,polynucleotide hybridization interactions, and so forth.

Non-specific binding is non-covalent binding between molecules that isrelatively independent of specific surface structures. Non-specificbinding may result from several factors including hydrophobicinteractions between molecules.

The methods of the present invention may be used to detect the presenceof a particular target analyte, for example, a nucleic acid,oligonucleotide, protein, enzyme, antibody or antigen. The methods mayalso be used to screen bioactive agents, i.e. drug candidates, forbinding to a particular target or to detect agents like pollutants.

The polyvalent ligand analytes will normally be poly(amino acids), i.e.,polypeptides and proteins, polysaccharides, nucleic acids, andcombinations thereof. Such combinations include components of bacteria,viruses, chromosomes, genes, mitochondria, nuclei, cell membranes andthe like.

For the most part, the polyepitopic ligand analytes to which the subjectinvention can be applied will have a molecular weight of at least about5,000, more usually at least about 10,000. In the poly(amino acid)category, the poly(amino acids) of interest will generally be from about5,000 to 5,000,000 molecular weight, more usually from about 20,000 to1,000,000 molecular weight; among the hormones of interest, themolecular weights will usually range from about 5,000 to 60,000molecular weight.

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.The analytes include drugs, metabolites, pesticides, pollutants, and thelike. Included among drugs of interest are the alkaloids. Among thealkaloids are morphine alkaloids, which includes morphine, codeine,heroin, dextromethorphan, their derivatives and metabolites; cocainealkaloids, which include cocaine and benzyl ecgonine, their derivativesand metabolites; ergot alkaloids, which include the diethylamide oflysergic acid; steroid alkaloids; iminazoyl alkaloids; quinazolinealkaloids; isoquinoline alkaloids; quinoline alkaloids, which includequinine and quinidine; diterpene alkaloids, their derivatives andmetabolites.

The term analyte further includes polynucleotide analytes such as thosepolynucleotides defined below. These include m-RNA, r-RNA, t-RNA, DNA,DNA-RNA duplexes, etc. The term analyte also includes receptors that arepolynucleotide binding agents, such as, for example, peptide nucleicacids (PNA), restriction enzymes, activators, repressors, nucleases,polymerases, histones, repair enzymes, chemotherapeutic agents, and thelike.

The analyte may be a molecule found directly in a sample such as a bodyfluid from a host. The sample can be examined directly or may bepretreated to render the analyte more readily detectible. Furthermore,the analyte of interest may be determined by detecting an agentprobative of the analyte of interest such as a specific binding pairmember complementary to the analyte of interest, whose presence will bedetected only when the analyte of interest is present in a sample. Thus,the agent probative of the analyte becomes the analyte that is detectedin an assay. The body fluid can be, for example, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, and the like.

The term “probe” or “probe molecule” refers to a molecule that binds toa target molecule for the analysis of the target. The probe or probemolecule is generally, but not necessarily, has a known molecularstructure or sequence. The probe or probe molecule is generally, but notnecessarily, attached to the solid substrate of the array. The probe orprobe molecule is typically a nucleotide, an oligonucleotide, or aprotein, including, for example, cDNA or pre-synthesized polynucleotidedeposited on the array. Probes molecules are biomolecules capable ofundergoing binding or molecular recognition events with targetmolecules. (In some references, the terms “target” and “probe” aredefined opposite to the definitions provided here.) The polynucleotideprobes require only the sequence information of genes, and thereby canexploit the genome sequences of an organism. In cDNA arrays, there couldbe cross-hybridization due to sequence homologies among members of agene family. Polynucleotide arrays can be specifically designed todifferentiate between highly homologous members of a gene family as wellas spliced forms of the same gene (exon-specific). Polynucleotide arraysof the embodiment of this invention could also be designed to allowdetection of mutations and single nucleotide polymorphism. A probe orprobe molecule can be a capture molecule.

The term “capture molecule” refers to a molecule that is immobilized ona surface. The capture molecule is generally, but not necessarily, bindsto a target or target molecule. The capture molecule is typically anucleotide, an oligonucleotide, or a protein, but could also be a smallmolecule, biomolecule, or nanomaterial such as but not necessarilylimited to a small molecule that is biologically active, nucleic acidsand their sequences, peptides and polypeptides, as well as nanostructurematerials chemically modified with biomolecules or small moleculescapable of binding to a target molecule that is bound to a probemolecule to form a complex of the capture molecule, target molecule andthe probe molecule. The capture molecule may be fluorescently labeledDNA or RNA. The capture molecule may or may not be capable of binding tojust the target molecule or just the probe molecule.

The term “molecule” generally refers to a macromolecule or polymer asdescribed herein. However, arrays comprising single molecules, asopposed to macromolecules or polymers, are also within the scope of theembodiments of the invention.

“Predefined region” or “spot” or “pad” refers to a localized area on asolid support. The spot could be intended to be used for formation of aselected molecule and is otherwise referred to herein in the alternativeas a “selected” region. The spot may have any convenient shape, e.g.,circular, rectangular, elliptical, wedge-shaped, etc. For the sake ofbrevity herein, “predefined regions” are sometimes referred to simply as“regions” or “spots.” In some embodiments, a predefined region and,therefore, the area upon which each distinct molecule is synthesized issmaller than about 1 cm² or less than 1 mm², and still more preferablyless than 0.5 mm². In most preferred embodiments the regions have anarea less than about 10,000 μm² or, more preferably, less than 100 μm²,and even more preferably less than 10 μm² or less than 1 μm².Additionally, multiple copies of the polymer will typically besynthesized within any preselected region. The number of copies can bein the hundreds to the millions. A spot could contain an electrode togenerate an electrochemical reagent, a working electrode to synthesize apolymer and a confinement electrode to confine the generatedelectrochemical reagent. The electrode to generate the electrochemicalreagent could be of any shape, including, for example, circular, flatdisk shaped and hemisphere shaped.

The term “nucleotide” includes deoxynucleotides and analogs thereof.These analogs are those molecules having some structural features incommon with a naturally occurring nucleotide such that when incorporatedinto a polynucleotide sequence, they allow hybridization with acomplementary polynucleotide in solution. Typically, these analogs arederived from naturally occurring nucleotides by replacing and/ormodifying the base, the ribose or the phosphodiester moiety. The changescan be tailor-made to stabilize or destabilize hybrid formation, or toenhance the specificity of hybridization with a complementarypolynucleotide sequence as desired, or to enhance stability of thepolynucleotide.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides, that comprise purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. Polynucleotides of the embodiments of theinvention include sequences of deoxyribopolynucleotide (DNA),ribopolynucleotide (RNA), or DNA copies of ribopolynucleotide (cDNA)which may be isolated from natural sources, recombinantly produced, orartificially synthesized. A further example of a polynucleotide of theembodiments of the invention may be polyamide polynucleotide (PNA). Thepolynucleotides and nucleic acids may exist as single-stranded ordouble-stranded. The backbone of the polynucleotide can comprise sugarsand phosphate groups, as may typically be found in RNA or DNA, ormodified or substituted sugar or phosphate groups. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. The polymers made of nucleotides such asnucleic acids, polynucleotides and polynucleotides may also be referredto herein as “nucleotide polymers.

An “oligonucleotide” is a polynucleotide having 2 to 20 nucleotides.Analogs also include protected and/or modified monomers as areconventionally used in polynucleotide synthesis. As one of skill in theart is well aware, polynucleotide synthesis uses a variety ofbase-protected nucleoside derivatives in which one or more of thenitrogens of the purine and pyrimidine moiety are protected by groupssuch as dimethoxytrityl, benzyl, tert-butyl, isobutyl and the like.

For instance, structural groups are optionally added to the ribose orbase of a nucleoside for incorporation into a polynucleotide, such as amethyl, propyl or allyl group at the 2′-O position on the ribose, or afluoro group which substitutes for the 2′-O group, or a bromo group onthe ribonucleoside base. 2′-O-methyloligoribonucleotides (2′-O-MeORNs)have a higher affinity for complementary polynucleotides (especiallyRNA) than their unmodified counterparts. Alternatively, deazapurines anddeazapyrimidines in which one or more N atoms of the purine orpyrimidine heterocyclic ring are replaced by C atoms can also be used.

The phosphodiester linkage, or “sugar-phosphate backbone” of thepolynucleotide can also be substituted or modified, for instance withmethyl phosphonates, O-methyl phosphates or phosphororthioates. Anotherexample of a polynucleotide comprising such modified linkages forpurposes of this disclosure includes “peptide polynucleotides” in whicha polyamide backbone is attached to polynucleotide bases, or modifiedpolynucleotide bases. Peptide polynucleotides which comprise a polyamidebackbone and the bases found in naturally occurring nucleotides arecommercially available.

Nucleotides with modified bases can also be used in the embodiments ofthe invention. Some examples of base modifications include2-aminoadenine, 5-methylcytosine, 5-(propyn-1-yl)cytosine,5-(propyn-1-yl)uracil, 5-bromouracil, 5-bromocytosine,hydroxymethylcytosine, methyluracil, hydroxymethyluracil, anddihydroxypentyluracil which can be incorporated into polynucleotides inorder to modify binding affinity for complementary polynucleotides.

Groups can also be linked to various positions on the nucleoside sugarring or on the purine or pyrimidine rings which may stabilize the duplexby electrostatic interactions with the negatively charged phosphatebackbone, or through interactions in the major and minor groves. Forexample, adenosine and guanosine nucleotides can be substituted at theN² position with an imidazolyl propyl group, increasing duplexstability. Universal base analogues such as 3-nitropyrrole and5-nitroindole can also be included. A variety of modifiedpolynucleotides suitable for use in the embodiments of the invention aredescribed in the literature.

When the macromolecule of interest is a peptide, the amino acids can beany amino acids, including α, β, or ω-amino acids. When the amino acidsare α-amino acids, either the L-optical isomer or the D-optical isomermay be used. Additionally, unnatural amino acids, for example,β-alanine, phenylglycine and homoarginine are also contemplated by theembodiments of the invention. These amino acids are well-known in theart.

A “peptide” is a polymer in which the monomers are amino acids and whichare joined together through amide bonds and alternatively referred to asa polypeptide. In the context of this specification it should beappreciated that the amino acids may be the L-optical isomer or theD-optical isomer. Peptides are two or more amino acid monomers long, andoften more than 20 amino acid monomers long.

A “protein” is a long polymer of amino acids linked via peptide bondsand which may be composed of two or more polypeptide chains. Morespecifically, the term “protein” refers to a molecule composed of one ormore chains of amino acids in a specific order; for example, the orderas determined by the base sequence of nucleotides in the gene coding forthe protein. Proteins are essential for the structure, function, andregulation of the body's cells, tissues, and organs, and each proteinhas unique functions. Examples are hormones, enzymes, and antibodies.

The term “sequence” refers to the particular ordering of monomers withina macromolecule and it may be referred to herein as the sequence of themacromolecule.

The term “hybridization” refers to the process in which twosingle-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.” For example, hybridization refers to theformation of hybrids between a probe polynucleotide (e.g., apolynucleotide of the invention which may include substitutions,deletion, and/or additions) and a specific target polynucleotide (e.g.,an analyte polynucleotide) wherein the probe preferentially hybridizesto the specific target polynucleotide and substantially does nothybridize to polynucleotides consisting of sequences which are notsubstantially complementary to the target polynucleotide. However, itwill be recognized by those of skill that the minimum length of apolynucleotide desired for specific hybridization to a targetpolynucleotide will depend on several factors: G/C content, positioningof mismatched bases (if any), degree of uniqueness of the sequence ascompared to the population of target polynucleotides, and chemicalnature of the polynucleotide (e.g., methylphosphonate backbone,phosphorothiolate, etc.), among others.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known in the art.

It is appreciated that the ability of two single strandedpolynucleotides to hybridize will depend upon factors such as theirdegree of complementarity as well as the stringency of the hybridizationreaction conditions.

As used herein, “stringency” refers to the conditions of a hybridizationreaction that influence the degree to which polynucleotides hybridize.Stringent conditions can be selected that allow polynucleotide duplexesto be distinguished based on their degree of mismatch. High stringencyis correlated with a lower probability for the formation of a duplexcontaining mismatched bases. Thus, the higher the stringency, thegreater the probability that two single-stranded polynucleotides,capable of forming a mismatched duplex, will remain single-stranded.Conversely, at lower stringency, the probability of formation of amismatched duplex is increased.

The appropriate stringency that will allow selection of aperfectly-matched duplex, compared to a duplex containing one or moremismatches (or that will allow selection of a particular mismatchedduplex compared to a duplex with a higher degree of mismatch) isgenerally determined empirically. Means for adjusting the stringency ofa hybridization reaction are well-known to those of skill in the art.

A “ligand” is a molecule that is recognized by a particular receptor.Examples of ligands that can be investigated by this invention include,but are not restricted to, agonists and antagonists for cell membranereceptors, toxins and venoms, viral epitopes, hormones, hormonereceptors, peptides, enzymes, enzyme converted substrates, cofactors,drugs (e.g. opiates, steroids, etc.), lectins, sugars, polynucleotides,nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

A “receptor” is molecule that has an affinity for a given ligand.Receptors may-be naturally-occurring or manmade molecules. Also, theycan be employed in their unaltered state or as aggregates with otherspecies. Receptors may be attached, covalently or noncovalently, to abinding member, either directly or via a specific binding substance.Examples of receptors which can be employed by this invention include,but are not restricted to, antibodies, cell membrane receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Receptorsare sometimes referred to in the art as anti-ligands. As the term“receptors” is used herein, no difference in meaning is intended. A“Ligand Receptor Pair” is formed when two macromolecules have combinedthrough molecular recognition to form a complex. Other examples ofreceptors which can be investigated by this invention include but arenot restricted to:

a) Microorganism receptors: Determination of ligands which bind toreceptors, such as specific transport proteins or enzymes essential tosurvival of microorganisms, is useful in developing a new class ofantibiotics. Of particular value would be antibiotics againstopportunistic fungi, protozoa, and those bacteria resistant to theantibiotics in current use.

b) Enzymes: For instance, one type of receptor is the binding site ofenzymes such as the enzymes responsible for cleaving neurotransmitters;determination of ligands which bind to certain receptors to modulate theaction of the enzymes which cleave the different neurotransmitters isuseful in the development of drugs which can be used in the treatment ofdisorders of neurotransmission.

c) Antibodies: For instance, the invention may be useful ininvestigating the ligand-binding site on the antibody molecule whichcombines with the epitope of an antigen of interest; determining asequence that mimics an antigenic epitope may lead to the-development ofvaccines of which the immunogen is based on one or more of suchsequences or lead to the development of related diagnostic agents orcompounds useful in therapeutic treatments such as for auto-immunediseases (e.g., by blocking the binding of the “anti-self” antibodies).

d) Nucleic Acids: Sequences of nucleic acids may be synthesized toestablish DNA or RNA binding sequences.

e) Catalytic Polypeptides: Polymers, preferably polypeptides, which arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products. Such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,which functionality is capable of chemically modifying the boundreactant.

f) Hormone receptors: Examples of hormones receptors include, e.g., thereceptors for insulin and growth hormone. Determination of the ligandswhich bind with high affinity to a receptor is useful in the developmentof, for example, an oral replacement of the daily injections whichdiabetics take to relieve the symptoms of diabetes. Other examples arethe vasoconstrictive hormone receptors; determination of those ligandswhich bind to a receptor may lead to the development of drugs to controlblood pressure.

g) Opiate receptors: Determination of ligands which bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

The term “fluid” used herein means an aggregate of matter that has thetendency to assume the shape of its container, for example a liquid orgas. Analytes in fluid form can include fluid suspensions and solutionsof solid particle analytes.

A “macromolecule” or “polymer” comprises two or more monomers covalentlyjoined. The monomers may be joined one at a time or in strings ofmultiple monomers, ordinarily known as “oligomers.” Thus, for example,one monomer and a string of five monomers may be joined to form amacromolecule or polymer of six monomers. Similarly, a string of fiftymonomers may be joined with a string of hundred monomers to form amacromolecule or polymer of one hundred and fifty monomers.

The term “biopolymer” is a polymer found in nature and includes, forexample, both linear and cyclic polymers of nucleic acids,polynucleotides, polynucleotides, polysaccharides, oligosaccharides,proteins, polypeptides, peptides, phospholipids and peptide nucleicacids (PNAs). The peptides include those peptides having either α-, β-,or ω-amino acids.

The term “Antigen” referers to the proteins that can be recognized(bound) by an antibody. Antigens are most commonly polypeptides orcarbohydrates, but they can also be lipids, nucleic acids, or even smallmolecules like neurotransmitters. A particular antibody molecule cantypically only interact with a small region of an antigen and in thecase of a polypeptide this is generally about 5-12 amino acids. Thisregion can be continuous or it can be distributed in different regionsof a primary structure that are brought together because of thesecondary or tertiary structure of the antigen. The region of an antigenthat is recognized by an antibody is called an epitope. In some cases,it is possible to make an antibody that is directed against the antigenbinding site of another antibody (i.e., the antigen binding site is theepitope). This type of antibody is called an anti-idiotype antibody. Inthis case, the antigen binding site of the anti-idiotype antibody can besimilar in structure to the original antigen (they both recognise thesame antibody.)

Embodiments of the invention relate to integrated chemiluminescencedevices and methods for monitoring molecular binding utilizing thesedevices and methods. These devices and methods can be used, for example,to identify antigen binding to antibodies. The devices include both achemiluminescence material and a detector integrated together.

More specifically, one embodiment is an integrated assay device. Thedevice includes a chemiluminescent material, a biopolymer, and a lightdetector. An activity of the biopolymer can activate thechemiluminescent material and the activity of the chemiluminescentmaterial can be detected by the light detector. Preferably, thebiopolymer is an antibody.

Preferably, the device further includes a support substrate, wherein thechemiluminescent material, the biopolymer; and the light detector are onthe support substrate. Preferably, the support substrate comprisesglass, nickel, magnetic metal, gold, silicon nitrocellulose, orpolyvinylidene difluoride (PVDF).

Preferably, the antibody includes a conjugate enzyme for activating thechemiluminescent material. A preferred conjugate enzyme is horseradishperoxidase. Preferably, the antibody is capable of activating thechemiluminescent material upon binding to an antigen. Preferably, theantibody is configured to bind to an antigen of interest.

Preferred chemiluminescent materials include diacylhydrazides.Alternatively, the chemiluminescent material may include luminol,N-(4-Aminobutyl)-N-ethylisoluminol, 4-Aminophthalhydrazide monohydrate,Bis(2-carbopentyloxy-3,5,6-trichlorophenyl) oxalate,9,10-Bis(phenylethynyl)anthracene, 5,12-Bis(phenylethynyl)naphthacene,2-Chloro-9,10-bis(phenylethynyl)anthracene,1,8-Dichloro-9,10-bis(phenylethynyl)anthracene, Lucifer Yellow CHdipotassium salt, Lucifer yellow VS dilithium salt, 85% (dye content),2,4,5-Triphenylimidazole, 9,10-Diphenylanthracene, Rubrene, orTetrakis(dimethylamino)ethylene.

Preferably, the light detector is a charge-coupled device (CCD) or acomplimentary metal-oxide semiconductor (CMOS).

Another embodiment is a method of making an integrated device. Themethod includes applying a chemiluminescent material to a supportsubstrate including a light detector, and applying a biopolymer to thesupport. Another embodiment of a method of making an integrated deviceincludes attaching a light detector to a support comprising achemiluminescent material and a biopolymer. Preferably, the biopolymeris an antibody.

Yet another embodiment is a method of detecting an antigen. The methodincludes introducing an analyte antigen to a device comprising achemiluminescent material, an antibody that binds to a target antigenand a light detector. The chemiluminescent material luminesces if theanalyte antigen is the target antigen.

Further aspects of the embodiments will be described in more detailswith reference to FIGS. 2 and 3. One embodiment of a preferred device isshown in FIG. 2 and includes a detector array and a chemiluminescentmaterial. A chemiluminescent material provides for chemiluminescencewhen activated. Preferred materials are cyclic diacylhydrazides such asluminol, which are activated by oxidation. Luminol is typicallyactivated by the horseradish peroxidase (HRP)/hydrogen peroxidecatalysed oxidation in alkaline conditions. Immediately followingoxidation, the luminol is in an excited state which then decays toground state via a light emitting pathway. Enhanced chemiluminescence ispreferably achieved by performing the oxidation of luminol by the HRP inthe presence of chemical enhancers such as phenols. This has the effectof increasing the light output approximately 1000 fold and extending thetime of light emission. The light produced by this enhancedchemiluminescent reaction peaks after 5-20 minutes and decays slowlythereafter with a half life of approximately 60 minutes.

Preferably the chemiluminescent material is applied to the same supportsubstrate as the detector. The support substrate can comprise multiplelayers and may include silicon, glass, silicon dioxide, transparentpolymers, 3D polymer gels, gold, and silver, etc. In this manner, anintegrated miniaturized assay device can be prepared in advance of assaytesting. An integrated device as used herein is a single device in whichthe component parts of the device are designed to be used together as asingle device and none of the components of the device are designed tobe used alone. Since the detector and the chemiluminescent material arepart of the same device, researchers can more easily and accuratelyperform assay analysis with little additional equipment.

Any light detector that is able to detect the chemiluminescence of thematerial can be used. Preferred detectors are CCD (charge-coupleddevice) and CMOS (complimentary metal-oxide semiconductor) sensors. BothCCD and CMOS detectors convert light produced by the chemiluminescentmaterial into electrons using an array of pixels. In a CCD detector, thecharge produced from the detector is actually transported across thechip and read at one corner of an array. An analog-to-digital convertercan then be used to turn each pixel's value into a digital value. Inmost CMOS devices, there are several transistors at each pixel thatamplify and move the charge using more traditional wires. The CMOSapproach may be more flexible because each pixel can be readindividually. Accordingly, a CMOS array can be used to detect severaldifferent antigens simultaneously as different parts of the array can beconfigured to detect different antigens.

Standard methods for producing CMOS arrays and CCD detectors can beutilized to produce the light detectors. The chemiluminescent materialcan then be dispersed in an inert polymer matrix and applied by spincoating or spray coating directly on top of the light detector substratewith functional groups for attachment of the antibody molecules. Thesecould be, for example, carboxylic acid groups for amine attachment ofantibodies. The thickness of the chemiluminescent material doped polymermatrix can be adjusted by spin coating speed. Antibody can be conjugatedto the polymer matrix via functional groups, for example, carboxylic oraldehyde groups.

Alternatively, the top of the CMOS detector substrate with functionalgroups can be coated with the chemiluminescent material and the antibodycan then be applied by spotting. The spotting is typically done bydipping a spotting needle into the antibody solution and bringing thespotting needle near contact with the substrate at locations where theantibodies are desired. The size of the antibody spot is determined bythe size of the spotting needle and the volume of antibody solutionretained by the spotting needle.

As shown in FIG. 2, an antibody that includes conjugate tag foractivating the chemiluminescent material is preferably applied onto thechemiluminescent material. For example an antibody that includes anoxidase enzyme conjugate can be used to activate a device that includesluminol. A preferred oxidase enzyme is HRP. Other preferred oxidaseenzymes include Myeloperoxidase, Glutathione Peroxidase, CholesterolOxidase f. Pseudomonas, Cholesterol Oxidase from Nocardia erythropolis,Choline Oxidase from Alcaligenes sp, D-Amino acid Oxidase from hogkidney, D-Amino acid Oxidase from hog kidney, Glycerol 3-phosphateOxidase from Aerococcus viridans, L-Amino acid Oxidase from Crotalusadamanteus, Lipoxidase from soybean, lyophilized, powder, SarcosineOxidase from Pseudomonas sp, and Xanthine Oxidase from buttermilk,lyophilized. Different antibodies may be applied to different parts ofthe support substrate to allow for the simultaneous detection ofdifferent antigens.

In an alternative embodiment, the chemiluminescent material and theantibody that includes a tag for activating the chemiluminescentmaterial can be applied to a support substrate. The support substratecan then be analyzed using a separate non integrated detector.

The operation of the device is shown in FIG. 3. In FIG. 3 an antigenattaches to the antibody in an exothermic reaction. The exothermicreaction provides the energy for the oxidase enzyme attached to theantibody to oxidize the chemiluminescent material. The luminesceneproduced by the chemiluminescent material can then be detected by thedetector.

Specifically, if the oxidase oxidizes the chemiluminescence material,light is emitted from the chemiluminescence material layer. The emittedlight is converted to electrons in the light detector, and the electronsare read out as electrical signal by the light detector. The electricalsignal can be further amplified or processed by a microprocessor or inan electrical circuit, or stored in a solid state device such as a flashmemory or a storage media like an optical disk or a hard drive.

In an alternative embodiment, the device utilizes a light source forstronger signal generation. The light source is positioned at an angleso that the direct illumination from the light source is not detected bythe CMOS or CCD detector. Preferred light sources include light emittingdiodes (LED), incandescent light bulbs, fluorescence light bulbs, gaslamps including mercury, argon, neon, and krypton lamps, and halogen andxenon lamps.

In another alternative embodiment, the light source can be positioneddirectly above the substrate. In this case, an optical filter may bepositioned between the chemiluminescence layer and the CMOS or CCD, toblock the light from the light source while transmitting light generatedby the oxidizing chemiluminescence material. Preferred optical filtersinclude thin film filters, for example, Bragg filters or any otherinterferenece filters, and absorption/reflection filters that absorb orreflect specific wavelength of light.

The device may also include micro-fluid channels for delivering a sampleto the device. Alternatively, samples may be applied to the devicethrough spotting techniques.

The devices and methods described herein can be used for a variety ofapplicants, for example, in the point of care and field devices fordiagnostics, forensic, pharmaceutical, agricultural, food inspection,biodefense, environmental monitoring, and industrial process monitoring.

This application discloses several numerical range limitations thatsupport any range within the disclosed numerical ranges even though aprecise range limitation is not stated verbatim in the specificationbecause the embodiments of the invention could be practiced throughoutthe disclosed numerical ranges. Finally, the entire disclosure of thepatents and publications referred in this application, if any, arehereby incorporated herein in entirety by reference.

1. An integrated assay device comprising in order: a light detectorhaving a light detector substrate; a layer of chemiluminescent material;and a biopolymer, wherein an activity of the biopolymer can activate thechemiluminescent material and the activity of the chemiluminescentmaterial can be detected by the light detector, wherein the layer ofchemiluminescent material is located directly on the light detectorsubstrate.
 2. The device of claim 1, wherein the biopolymer is anantibody.
 3. The device of claim 1, further comprising a supportsubstrate, wherein the chemiluminescent material, the biopolymer; andthe light detector are on the support substrate.
 4. The device of claim2, wherein the support substrate comprises glass, nickel, magneticmetal, gold, silicon, nitrocellulose, or polyvinylidene difluoride(PVDF).
 5. The device of claim 2, wherein the antibody comprises aconjugate enzyme for activating the chemiluminescent material.
 6. Thedevice of claim 5, wherein the conjugate enzyme is horseradishperoxidase.
 7. The device of claim 2, wherein the antibody is capable ofactivating the chemiluminescent material upon binding to an antigen. 8.The device of claim 2, wherein the antibody is configured to bind to anantigen of interest.
 9. The device of claim 1, wherein thechemiluminescent material comprises a cyclic diacylhydrazides.
 10. Thedevice of claim 1, wherein the chemiluminescent material comprises amaterial selected from the group consisting of luminol,N-(4-Aminobutyl)-N-ethylisoluminol, 4-Aminophthalhydrazide monohydrate,Bis(2-carbopentyloxy-3,5,6-trichlorophenyl)oxalate,9,10-Bis(phenylethynyl)anthracene, 5,12-Bis(phenylethynyl)naphthacene,2-Chloro-9,10-bis(phenylethynyl)anthracene,1,8-Dichloro-9,10-bis(phenylethynyl)anthracene, Lucifer Yellow CHdipotassium salt, Lucifer yellow VS dilithium salt, 85% (dye content),2,4,5-Triphenylimidazole, 9,10-Diphenylanthracene, Rubrene, andTetrakis(dimethylamino)ethylene.
 11. The device of claim 1, wherein thelight detector is a charge-coupled device (CCD) or a complimentarymetal-oxide semiconductor (CMOS).